We found that the overexpression of a macrophage signature in expression-profile studies of diagnostic lymph-node specimens obtained from patients with classic Hodgkin's lymphoma was associated with the failure of primary treatment. Using immunohistochemical analysis, we also found that an increased number of CD68+ cells (a marker of benign macrophages) in the diagnostic sample was associated with a poorer outcome in an independent set of samples from 166 patients. Multivariate analysis revealed that the number of CD68+ cells was also associated with the outcome of secondary treatment, independently of the International Prognostic Score.
Three previous studies that have used expression profiles of the microenvironment in Hodgkin's lymphoma have been reported.23,24,26
Of these, one identified a gene signature of macrophages but did not show the clinical value of tumor-associated macrophages assessed by means of a common immunohistochemical marker.23
The association between the number of macrophages and treatment outcome has also been studied in other B-cell cancers.19,27,28
Differences in survival among patients with various lymphoma subtypes, which are linked to macrophage content, might be explained by the variable presence of macrophages with M1 or M2 differentiation in biopsy samples, indicating distinct biologic features of the tumors.29
However, in these lymphoma subtypes, including Hodgkin's lymphoma, the functional link between macrophage numbers and the contribution of these cells to the treatment outcome remains unclear.23,30
Our gene-expression classifier for the outcome of primary treatment outcome revealed MMP11
, a gene that has been found by other investigators to be expressed in tumor-associated macrophages involved in remodeling of apoptotic lymphatic tissue.25
Using immunohistochemical analysis, we were able to confirm the correlation between the number of MMP11+ cells and progression-free survival in an independent cohort of patients. However, MMP11 stained many different cell types, including macrophages, and thus did not allow us to identify the particular cells that were responsible for the production of the protein.
Our findings also validate the recent report of a correlation between an increased number of small B cells and a favorable outcome.26
The recently described correlation between the number of CD20-positive B cells and survival in patients with classic Hodgkin's lymphoma31
needs to be reassessed in the context of clinical studies showing successful treatment with the addition of rituximab to standard chemotherapy.32,33
We report the clinical value of a single marker, CD68, in the identification of tumor-associated macrophages by immunohistochemical analysis, an analytic method that can be easily incorporated into a routine diagnostic approach. The use of such markers in combination with well-established clinical risk factors could improve on the predictive value of a single biomarker used alone.
We focused on tumor-associated macrophages because of the strong signal from the gene-expression data and the recently renewed interest in these non-neoplastic cells as major contributors to the biologic features of lymphoma and outcome prediction.19
The value of assessing the number of tumor-associated macrophages as a biomarker is highlighted by the association between these cells and the outcome after secondary therapy with autologous stem-cell transplantation, a widely used treatment option. Accurate prediction of the outcome after secondary treatments with curative intent would provide better risk stratification for these therapeutic options. Clinical predictors of the outcome after autologous stem-cell transplantation have been of limited value.34
In addition, our finding that in the tissue-microarray cohort, none of the patients with limited-stage disease and a low number of macrophages died is encouraging, since the applicability of the International Prognostic Score is restricted to advanced-stage disease. Thus, the CD68+ macrophage content represents a biomarker with clinical applicability in all stages of classic Hodgkin's lymphoma, both at the time of diagnosis and at the time of relapse.
In summary, our study showed the value of enumerating CD68+ macrophages in diagnostic lymph-node samples for prediction of the outcome after primary treatment and secondary treatment (in particular, autologous stem-cell transplantation). The absence of an increased number of CD68+ cells in patients with limited-stage disease defines a subgroup of patients for whom the rate of long-term disease-specific survival is 100% with the use of available treatments.